Characterization of genes encoding diterpene cyclases responsible for phytoalexin biosynthesis in rice.

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From rice 14 diterpenoid phytoalexins were isolated; momilactones, phytocassanes, and oryzalexins. We have isolated cDNAs encoding diterpene cyclases responsible for biosynthesis of these phytoalexins; OsCPS2, OsCPS4, OsKSL4, OsKSL7, OsKSL8, OsKSL10. This project was for characterization of the cyclase genes. 1) Gene expression patterns: The expression levels in roots of OsCPS2, OsCPS4, OsKSL4 OsKSL7, and OsKSL8, were higher than those in shoots. The expression levels of OsCPS2, OsCPS4, OsKSL4, and OsKSL7 were drastically increased in the rice fruits from heading stage to flowering stage. Transient assay using green fluorescence protein suggested that these diterpene cyclases are transported thin plastids. 2) Isolation of their homologues: From rice KS like homologue cDNAs, OsKSL5 and OsKSL6, which encode ent-pimara-8,15-diene synthase and ent-kaur-15-ene synthase respectively, were isolated. Two homologue cDNAs of OsCPS2 and OsCPS4 were isolated from wheat, other species in Poaceae. 3) Relationship of rice diterpene phytoalexins with pathogenic tolerance: The expression levels of six genes increased after infection by Magnaporthe grisea. We did not succeed in screening Tos 17-insertion-knock-out mutants and production of knock-down mutants by RNAi. 4) Comparison of OsCPS2 to OsCPS1 that is responsible for gibberellin biosynthesis: The severe dwarf phenotype of mutants lacking in OsCPS1 were rescued by ectopic expression of OsCPS2. This result indicated that regulation of OsCPS1 expression is different from that of OsCPS2 expression. The basic enzymatic properties of OsCPS2 were almost same as those of OsCPS1, such as divalent cation requirement, optimal pH, optimal temperature and kinetic parameter. However, the activity of OsCPS2 was suppressed by both of substrate geranylgeranyl diphosphate and a gibberellin-biosynthesis-inhibitor AMO 1618 less than that of OsCPS1.